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H Kleinkauf

Publications and source records attributed to H Kleinkauf.

At least 19 recordsLinked to original sources
PubMed1996

Biosynthesis of acylpeptidolactones of the daptomycin type. A comparative analysis of peptide synthetases forming A21978C and A54145.

A21978C and A54145 are antibacterial 13-residue peptides with a medium-chain-acylated amino terminus and a 10-residue lactone ring; they are produced by strains of Streptomyces roseosporus and Streptomyces fradiae, respectively. The structural differences in their peptide chains, which include amino acid replacements and modifications (L-Glu2-->L-Asn, L-Asn(OH)3-->L-Asp, Sar5-->Gly, L-Ala6-->L-Orn, L-Lys8-->D-Ala, L-Asp(OMe)9-->L-Asp, L-Asn11-->D-Ser, and L-lle13-->L-Kyn; Sar = sarcosine; L-Orn = L-ornithine, L-Kyn = L-kynurenine), reside in the multienzymatic templates directing their biosynthesis. We have examined the peptide synthetases employing immunodetection and substrate activation detected by the amino-acid-dependent ATP-PP1-exchange reaction. Two different antibodies specific for actinomycin synthetase 2 and a peptide sequence characteristic of acyl-CoA-synthetases/peptide synthetases were applied. For the A21978 system two peptide synthetases of 670 and 240 kDa were detected, together with two similar proteins of 630 and 440 kDa occurring in varying amounts. The latter are suggested to be degradation products of an unstable multienzyme. Activation of L-Asp, L-Thr, Gly, L-Orn, L-Ala and L-Ser were assigned to the high-molecular-mass components of 670, 630 and 440 kDa. The 240-kDa protein was purified to homogeneity and shown to catalyse activation of L-kynurenine. The A54145 system consisted of three peptide synthetases of 690, 590 and 250 kDa. Activations of L-Asn. L-Thr and Gly were found. The 250-kDa synthetase was capable of activating isoleucine and valine. Both systems thus show a comparable organisation; implications for the modular construction of their peptide synthetases are discussed.

Amino Acid Sequence↗
PubMed1996

A nonribosomal system of peptide biosynthesis.

This review covers peptide structures originating from the concerted action of enzyme systems without the direct participation of nucleic acids. Biosynthesis proceeds by formation of linear peptidyl intermediates which may be enzymatically modified as well as transformed into specific cyclic structures. The respective enzyme systems are constructed of biosynthetic modules integrated into multienzyme structures. Genetic and DNA-sequence analysis of biosynthetic gene clusters have revealed extensive similarities between prokaryotic and eukaryotic systems, conserved principles of organisation, and a unique mechanism of transport of intermediates during elongation and modification steps involving 4'-phospho-pantetheine. These similarities permit the identification of peptide synthetases and related aminoacyl-ligases and acyl-ligases from sequence data. Similarities to other biosynthetic systems involved in the assembly of polyketide metabolites are discussed.

Amino Acid Sequence↗
PubMed1996

Reversible denaturation of cyclosporin synthetase by urea.

The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo-(D-alanyl-N-methylleucyl), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifuntional polypeptide.

Cyclosporine↗
PubMed1995

Characterization of tyrocidine synthetase 1 (TY1): requirement of posttranslational modification for peptide biosynthesis.

Tyrocidine synthetase 1 (TY1), produced by Bacillus brevis ATCC 8185, consists of a single multifunctional polypeptide chain catalyzing the activation, thioesterification, and epimerization of phenylalanine. Because we were concerned about possible posttranslational issues, a comparative study between the wild-type isolate and the in Escherichia coli overexpressed protein was performed. Analysis by matrix assisted laser desorption mass spectrometry (MALDI) provided a molecular mass of 122,516 +/- 120 Da for the recombinant protein, which is in agreement with the value of 122,590 Da calculated from the gene sequence. MALDI analysis of the tryptic fragments revealed that in the recombinant TY1 the putative 4'-phosphopantetheine binding site (562Ser) is not modified by the cofactor. The substrate specificity profiles of the amino acid dependent ATP[32P]PPi exchange reactions were identical, including activation of L-phenylserine, L-tyrosine, and L-methionine. However, the rates of the reverse adenylation reaction for the recombinant protein were only 22% relative to those of the wild-type enzyme. The aminoacylation levels of about 60% for TY1 from Bacillus brevis reduced to 1.4% in the overexpressed protein. A similar distribution of the D- and the L-isomer was detected at the thioester attachment site. The pI values of the wild-type and expressed TY1 are 4.9 and 5.0, respectively. In conclusion, it could be established that apo- and holo-TY1 differ in their amino acid activating properties. Posttranslational modification by 4'-phosphopantetheine is an essential requirement for aminoacylation, epimerization, and thus the functioning of the multienzyme in peptide synthesis.

Adenosine Triphosphate↗
PubMed1995

Expression of an active adenylate-forming domain of peptide synthetases corresponding to acyl-CoA-synthetases.

Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1,629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32P]PP(i) exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.

Adenosine Monophosphate↗
PubMed1995

The nonribosomal peptide biosynthetic system--on the origins of structural diversity of peptides, cyclopeptides and related compounds.

A variety of peptides have been detected in microorganisms. Some have found applications in various fields, for example the classical beta-lactam antibiotics, immunosuppressors like cyclosporin, promising new antibacterials like teichoplanin or daptomycin and antifungals like echinocandin. For none of these has it been established how their complicated biosynthetic pathways have evolved or what functions they fulfill within or for their producers. So it is unclear what selection processes limit the range of their structural analogues within various groups of microorganisms. We here consider recent data in the field of biosynthesis and how they may suggest mechanisms of genetic diversity. These may illustrate the complexity of genetic and intracellular organization of biosynthetic pathways and indicate the cellular context of some metabolites related to the complex background of the production of each metabolite. Research focusing on various targets like the increase of productivity of fermentations or the spread of resistances to antibacterials is slowly being understood.

Amino Acid Sequence↗
PubMed1994

Identification of the ATP binding site in tyrocidine synthetase 1 by selective modification with fluorescein 5'-isothiocyanate.

Identification of the nucleotide binding site in peptide synthetases has been approached by affinity labeling of tyrocidine synthetase 1 with fluorescein 5'-isothiocyanate. Binding was accompanied by irreversible inhibition of the ATP-dependent phenylalanine activation reaction and was prevented in the presence of MgATP2-. The reaction obeyed pseudo first-order rate kinetics and was accelerated by Mg2+. Complete inhibition corresponded to incorporation of 2.3 mol of fluorescein 5'-isothiocyanate (FITC)/mol of protein. Upon protection by MgATP2-, about 1 mol of FITC is still incorporated; however, this does not affect activity. The modified synthetase was extensively fragmented by tryptic digestion and the labeled fragments isolated by reverse-phase high performance liquid chromatography. Two peptides, DHQVKIR and LDKMPLTPNDKIDR, have been identified by sequencing, and the FITC conjugate of the former peptide has been detected by laser desorption mass spectrometry. The labeled residues, Lys-422 and Lys-505, are located within highly conserved segments of this new class of synthetases.

Adenosine Triphosphate↗
PubMed1994

ATP binding in peptide synthetases: determination of contact sites of the adenine moiety by photoaffinity labeling of tyrocidine synthetase 1 with 2-azidoadenosine triphosphate.

Characterization of the nucleotide binding domain in peptide synthetases was approached by photoaffinity labeling of tyrocidine synthetase 1 (TY1) with 2-azidoadenosine triphosphate (2-azido-ATP). Exposure of TY1 in the presence of photolabel to irradiation with ultraviolet light resulted in a time-dependent covalent modification of the enzyme with a concomitant loss of catalytic activity. Inactivation was not observed if incubation was performed in the absence of either light or the nucleotide analogue. Specificity of labeling was indicated by the ability of 2-azido-ATP to serve as a substrate in the amino acid activation reaction. The modified protein was subjected to tryptic digestion, and the fragments labeled by the nucleotide analogue were purified by reverse-phase high-performance liquid chromatography. Sequence analysis identified three tryptic peptides corresponding to residues G373-K384, W405-R416, and L483-K494, derived from the N-terminal half of the TY1 sequence. As this region shows similarity to strongly conserved regions in other peptide synthetases and acyl-CoA synthetases, it is considered to be the region catalyzing aminoacyl adenylate formation. The identified sequences appear to define components of the nucleotide binding domain found in close proximity to the adenine ring in ATP. Conservation of primary structure and homology to other carboxyl-activating enzymes of this superfamily, including peptide synthetases, insect luciferases, and acyl-CoA synthetases, is discussed.

Adenine↗
PubMed1994

Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis.

A specific alanine racemase, which is a key enzyme in the biosynthesis of the undecapeptide cyclosporin A, was purified to electrophoretic homogeneity from the fungus Tolypocladium niveum. This is the first enzyme of this kind isolated from a eucaryotic organism. The enzyme catalyzes the reversible racemization of alanine and requires pyridoxal phosphate as the exclusive cofactor. Km values for L- and D-alanine were found to be 38 and 2 mM, respectively. Maximal reaction velocity was observed at 42 degrees C and pH 8.8 for the L to D direction. Molecular mass determinations of the denatured enzyme by SDS-polyacrylamide gel electrophoresis gave a value of 37 kDa, whereas gel filtration calibration studies yielded a value between 120 and 150 kDa, indicating an oligomeric native structure.

Alanine Racemase↗
PubMed1994

Mechanism of cyclosporin A biosynthesis. Evidence for synthesis via a single linear undecapeptide precursor.

Cyclosporin A is synthesized by cyclosporin synthetase, a multienzyme polypeptide. This enzyme catalyzes at least 40 reaction steps in an assembly belt-like mechanism. It activates all constituent amino acids of cyclosporin A to thioesters via amino acyladenylates and carries out specific N-methylation reactions. During elongation, the activated amino acids are linked by peptide bonds leading to enzyme-bound nascent peptide chains. Some of the linear peptides of the growing cyclosporin A chain were isolated and their N-terminal amino acid was determined. D-Alanine at position 8 of the cyclosporin A molecule was found to be a starting amino acid in the biosynthetic process of cyclosporin A formation. Four intermediate peptides of the growing peptide chain of cyclosporin A could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. That these intermediate peptides were bound by thioester linkage to cyclosporin synthetase could be demonstrated by liberation of the peptides with performic acid. The peptides strongly suggest the stepwise synthesis of a single linear peptide precursor of cyclosporin A.

2,2'-Dipyridyl↗
PubMed1993

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, the multienzyme integrating the four primary reactions in beta-lactam biosynthesis, as a model peptide synthetase.

ACV synthetase forms the tripeptide precursor of penicillins and cephalosporins from alpha-aminoadipate, cysteine, and valine. Catalytic sites for substrate carboxyl activation as adenylates, peptide bond formations, epimerization and release of the tripeptide-thioester are integrated in multifunctional enzymes of 405 to 425 kD. These have been characterized from several pro- and eukaryotic beta-lactam producers. Implications of these results for the thio-template mechanism of peptide formation are discussed, as well as the use of this multienzyme as a model system for enzymatic peptide synthesis.

Amino Acid Sequence↗
PubMed1992

Two new cyclophilins from Fusarium sambucinum and Aspergillus niger: resistance of cyclophilin/cyclosporin A complexes against proteolysis.

Two new peptidyl-prolyl-cis/trans-isomerases were purified to homogeneity from Fusarium sambucinum and Aspergillus niger. They belong to the class of cyclosporin A binding proteins (cyclophilins) and have molecular masses of about 18 kDa. As has been shown for other cyclophilins, the isomerase activity of the enzymes is inhibited by cyclosporin A in the nanomolar range. Furthermore binding of cyclosporin A prevents proteolytic digestion of the cyclophilin/cyclosporin complexes by the endoproteases GluC, LysC and alpha-chymotrypsin, in contrast to the free cyclophilins, which are readily cleaved by these proteases. We could also observe this protection for cyclophilins from sheep thymus and from the cyclosporin producing fungus Tolypocladium inflatum.

Amino Acid Isomerases↗
PubMed1992

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide. Re-evaluation of the molecular mass of various peptide synthetases.

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re-evaluated by SDS-PAGE and CsCl density gradient centrifugation. In SDS-PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremely low protein concentrations, such as 10-50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re-evaluated in SDS-PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.

Cesium↗
PubMed1992

A highly specific D-hydroxyisovalerate dehydrogenase from the enniatin producer Fusarium sambucinum.

A highly specific D-hydroxyisovalerate (D-HIV) dehydrogenase, which is a key enzyme in depsipeptide synthesis, was purified to near homogeneity from the enniatin-producing fungus Fusarium sambucinum. The enzyme catalyzes the reversible reaction of 2-ketoisovalerate (2-KIV) to D-HIV. It is strictly dependent on NADPH and exhibits a high substrate specificity with respect to 2-KIV. NADH was not accepted by the enzyme. Km values for 2-KIV and NADPH were found to be 200 and 333 microM, respectively. D-HIV dehydrogenase consists of a single polypeptide chain with a molecular mass of about 53 kDa. Optimum temperature for the reduction of 2-KIV was 35 degrees C and for the oxidation reaction was 45 degrees C. The optimum pH was found to be 7 for the reduction and 8-9 for the oxidation reaction.

Alcohol Oxidoreductases↗
PubMed1992

Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.

Amino Acyl-tRNA Synthetases↗
PubMed1992

[Biosynthesis of peptides: a non-ribosomal system].

The biosynthesis of peptides in nonribosomal systems is accomplished by complex multienzymes. These multienzymes assemble the required template for the construction of each natural product in the form of linearly coupled modules. This organization principle permits the integration of multistep synthetic processes on a single macromolecule.

Amino Acid Sequence↗
PubMed1992

Enzymatic biosynthesis of cyclosporin A and analogues.

The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1,400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.

Amino Acid Sequence↗